Method for isolating highly pure nucleic acid with magnetic particles

ABSTRACT

The invention also relates to use of an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, as a washing solution for washing magnetic particles with nucleic acid adsorbed thereto.

The invention relates to methods for isolating nucleic acid from asample solution, wherein the nucleic acid is separated from the samplesolution by adsorption to magnetic particles, followed by separation ofthe magnetic particles with the nucleic acid adsorbed thereto from thesample solution by application of a magnetic field. Subject of theinvention are also uses of washing buffers in such methods.

STATE OF THE ART

Obtaining DNA or RNA sufficiently free of contaminants for molecularbiological applications is difficult in view of the complex systems inwhich the DNA or RNA is typically found. These systems, such asbiological samples, such as tissue, cells from body fluids such asblood, cultured cells, or artificial systems such as agarose gels or PCRreaction samples, typically include significant quantities ofcontaminants from which the DNA or RNA of interest must be separatedbefore being used in a molecular biological procedure.

Conventional protocols for obtaining DNA or RNA from cells entailsuspending the cells in a solution and using enzymes and/or chemicals,to lyse the cells, thereby releasing the nucleic acid contained withinthe cells into the resulting lysate solution. For isolation of RNA, theconventional lysis and solubilization procedures that are applied forreleasing DNA include additional measures for inhibition ofribonucleases and contaminants to be separated from the RNA includingDNA.

Silica materials, including glass particles, such as glass powder,silica particles, and glass microfibers prepared by grinding glass fiberfilter papers, and including diatomaceous earth, are used in the art incombination for example with aqueous solutions of chaotropic salts toseparate DNA from other substances and render the DNA suitable for usein molecular biological procedures.

Glass particles, silica particles, silica gel, and mixtures of the abovehave been configured in various different forms to produce matricescapable of reversibly binding nucleic acid materials by adsorption tothe surface when placed in contact with a medium containing suchmaterials in the presence of binding inducing agents like chaotropicagents. Such matrices are designed to remain adsorbed to the nucleicacid material while the matrix is exposed to an external force such ascentrifugation or vacuum filtration to separate the matrix and nucleicacid material adsorbed thereto from the remaining media components. Thenucleic acid material usually is then eluted from the matrix by exposingthe matrix to an elution solution, such as water or an elution buffer.Numerous commercial sources offer silica-based matrices designed for usein centrifugation and/or filtration isolation systems (e.g. theQiaPrep®, QIAamp® and AllPrep® lines of nucleic acid isolation systemsfrom QIAGEN, Hilden, Germany).

Magnetically responsive particles (herein referred to as ‘magneticparticles’) and methods for using them have been developed for theisolation of nucleic acid materials. Several different types of magneticparticles designed for use in nucleic acid isolation are described inthe literature and are available from commercial sources. Such magneticparticles generally fall into either of two categories, those designedto reversibly bind nucleic acid materials directly, and those designedto do so indirectly, i.e. through at least one intermediary substance.The intermediary substance is referred to herein as a ‘label’. Forexample, one such commonly employed label, biotinylated oligonucleotidedeoxythymidine (oligo-dT), forms hydrogen bonds with the polyadenosinetails of mRNA molecules in a medium.

Magnetic particles which bind nucleic acids directly are often based onsilica, such as magnetically responsive siliceous-oxide coated particles(also referred to as magnetic beads; e.g. available under the trademarksMagAttract® from QIAGEN, Hilden, Germany; or MagneSil® from Promega,Madison, USA). Nucleic acids adhere to these particles in the presenceof binding inducing agent like a chaotropic salt, e.g. guanidiniumhydrochloride or guanidinium isothiocynate, alone or in combination witha binding additive, like an alcohol; e.g. ethanol. After separation ofthe particles, if desired, elution of the adsorbed nucleic acids fromthe particles may be achieved easily by incubation of the particles inwater or a buffer with low ionic strength.

Several different methods of automated separation of magnetic particlesare known from the art. One method is to insert a magnetic ormagnetizable device, such as a rod, into the medium containing themagnetic particles, binding the magnetic particles to the magnetic ormagnetizable device, and removing the magnetic or magnetizable device.Another method is to bring a magnetic or magnetizable device intospatial proximity to the container which contains the medium and themagnetic particles. The magnetic particles bind to the container walland the medium can be removed without carry-over of magnetic particles.

WO 01/71732 A2 discloses methods for producing magnetic particles basedon silica and iron oxide and their use in isolating biologicalmolecules. A molecule of interest, such as nucleic acids like DNA and/orRNA, is adhered to the magnetic particles, usually followed byseparation of the magnetic particles from the solution, washing andelution of the molecules. Binding of nucleic acids to the magneticparticles is preferably carried out in an aqueous solution comprising ahigh concentration of chaotropic salt and/or a low molecular weightalcohol, such as isopropanol or ethanol. Elution of the nucleic acidfrom the magnetic particles is preferably carried out with a low saltbuffer. In such a process, the desired molecule, such as the nucleicacid, usually is desired to be obtained in relatively pure form. Thus,the chaotropic salt from the starting solution needs to be removed asmuch as possible. In order to achieve this, washing steps typically arecarried out with one or more buffers preferably having a lower saltconcentration than the starting solution. In order to maintain bindingconditions in the multiple washing steps, the buffers generally compriserelatively high levels of ethanol, such as 70% (w/w). In a final washingstep, pure ethanol may be used. After the final washing step it iscommon that the sample with magnetic particles and the molecule boundthereto is left standing at room temperature to dry, such that ethanolis removed by volatilization. However, in view of the high saltconcentration of the starting material, and since washing buffersusually comprise a salt, the nucleic acid obtained according to such amethod may still comprise residual salt. Further, evaporation of ethanolby drying is relatively time-consuming. In a standard process,evaporation often does not fully dry the sample and deplete itcompletely from ethanol. Additional washing steps and extensive dryingmay further increase the purity, but such a process would be even moretime consuming and not be convenient any more for routine use.

WO 03/091452 A1 discloses a method for isolating nucleic acid frombiological materials with magnetic particles. In order to preventaggregate formation of the magnetic particles, specific washingsolutions are used comprising lower alcohols. Further, the washingsolutions may comprise chaotropic salts in relatively high amounts.Thus, the problem remains that the final nucleic acid product maycomprise undesired salt or alcohol impurities.

WO 2005/021748 A1 discloses a further method for isolating biopolymersfrom aqueous solution with magnetic particles. The biopolymers are boundto magnetic particles from aqueous solution comprising salt and ethanol,followed by washing and elution. In order to improve the handling of themagnetic particles, an additive, such as polyethylene glycol is added.Washing steps are carried out with washing solutions comprisingrelatively high amounts of chaotropic salt and ethanol, followed bydrying for ethanol removal.

Commercial kits for isolating nucleic acids from various sources and ofvarious sizes with magnetic particles are for instance available fromQiagen, Hilden, Germany, under the trademark MagAttract®. The basicprotocol, which is for example used in the MagAttract® HMW DNA Kit, issimilar as described further above and comprises steps of binding DNAfrom aqueous solution in the presence of chaotropic salts and ethanol,followed by washing steps and final elution of DNA. The standard washingsteps are carried out in the presence of relatively high saltconcentration and ethanol. However, in order to remove salt and ethanolimpurities which might interfere with subsequent downstream processesfrom the DNA product a final washing step is included, in which thepellet of magnetic particles with DNA bound thereto, when fixed to thewall of the sample tube by magnetic force, is just briefly rinsed withwater. When proceeding accordingly, the water mostly contacts only thesurface of the pellet adhered to the sample tube wall. According to thehandbook, it is important that the water is not added directly onto theparticles, but carefully pipetted into the sample tube against the sidefacing away from the magnetic particle pellet with bound DNA. Further,all pipetting steps shall be performed carefully to avoid disturbing thefixed magnetic particle pellet. These precautions are considerednecessary because the rinsing step is in fact carried out undernon-binding conditions, under which the nucleic acid is normally elutedfrom the particles. Thus, it is expected that intensive washing resultsin a loss of nucleic acids. Accordingly, in the same protocol it isnoted that pure water can be used as the elution buffer. However, sincethe magnetic particle pellet, which is fixed to the tube wall bymagnetic force, is not disaggregated under such a rather superficialwashing procedure, and since this water rinsing is only applied for ashort time only, a portion of the salt and/or ethanol may remain trappedin the magnetic particle pellet and, thereby, cannot be removed. Thus,it is desired to further improve the purity of the finally purified andisolated nucleic acid, either in pure form or dissolved.

US2009/0191566 A1 discloses a method for isolating biopolymers fromaqueous solution with magnetic particles by affinity binding. Themagnetic beads are modified with a first and a second functional group.Specifically, the functional groups are affinity ligands such asoligo-dT or streptavidin, which specifically bind target molecules suchas mRNA or biotin-labelled DNA. As is common practice in the state ofthe art, the wash buffer must have a “sufficiently high saltconcentration”, such that “the nucleic acid does not elute off the solidphase carrier, but remains bound to the microparticles”. Consequently,usually the salt has to be removed in a subsequent purification step toavoid impairment in downstream applications.

Hawkins et al, Nucl. Acids Res. 1994, Vol. 22, Nr. 21, 4543-4544,discloses a method for DNA purification with carboxyl-coated magneticbeads. The document discloses a final wash step using a comparativelylow salt concentration. However, the factual salt concentration in saidfinal washing step actually is relatively high due to the remainders ofa preceding wash step with a quite high salt concentration of 5M NaCl.Overall, the process is inefficient because the washing step reduces theyield to about 80%. Therefore, the method is not suitable forquantitative nucleic acid isolation.

WO2004/090132 A2 discloses a method for isolating genomic nucleic acidwith solid phase carriers comprising functional groups for binding thenucleic acids. Specifically, magnetic beads are used which are modifiedwith carboxyl groups. The disclosed properties of the wash buffers arebasically the same as disclosed in US2009/0191566A1 as discussed above.Again, the wash buffer should have a sufficiently high saltconcentration, such that the nucleic acid remains bound to themicroparticles and as a consequence the salt usually will have to beremoved in a subsequent purification step.

WO99/58664 describes a similar process for isolating nucleic acids withcarboxyl-coated magnetic beads. Suitable buffers should have propertiesand relatively high salt concentration as described above forUS2009/0191566A1 or WO2004/090132, in order to ensure that nucleic acidsremain bound to the microparticles.

Overall, there is a continuous need in the prior art for a simple andefficient process for isolating nucleic acids with magnetic particles,in which the level of impurities in the finally isolated nucleic acid,in particular DNA, is low.

PROBLEM UNDERLYING THE INVENTION

The problem underlying the invention is to provide a method forisolating nucleic acid from a sample solution, which overcomes theabove-mentioned drawbacks. Specifically, the problem underlying theinvention is to provide an improved method for isolating nucleic acid,in which the finally isolated nucleic acid has a high purity, whether inpure form or dissolved. The nucleic acid when not obtained in pure formbut as an isolation product should basically contain, preferably evenconsist of the elution buffer and the desired nucleic acid. The level ofimpurities, such as salt and organic solvent, such as alcohol likeethanol, should be low.

It is a further problem underlying the invention to provide a simplemethod for isolating highly pure nucleic acid. The process shallcomprise only a small number of process steps, which should be carriedout relatively easily and conveniently. Thus, the method shall beapplicable for routine use in standard laboratory practice as well as inautomated form, i.e. with robots and work stations. The method shall beavailable with standard laboratory materials and devices.

DISCLOSURE OF THE INVENTION

Surprisingly, it was found that the problem underlying the invention isovercome by methods according to the claims. Further embodiments of theinvention are outlined throughout the description.

Subject of the invention is a method for isolating nucleic acid from asample solution, wherein the nucleic acid is separated from the samplesolution by adsorption to magnetic particles, followed by separation ofthe magnetic particles with the nucleic acid adsorbed thereto from thesample solution by application of a magnetic field, followed by at leastone washing step (w), comprising

-   -   (w1) suspending the magnetic particles with the nucleic acid        adsorbed thereto in a washing solution, which is an aqueous        solution, which has a total salt concentration below 100 mM and        does not comprise an organic solvent,    -   (w2) incubating the magnetic particles with the nucleic acid        adsorbed thereto in the washing solution, and    -   (w3) separating the magnetic particles with the nucleic acid        adsorbed thereto from the washing solution.

The inventive method is directed in particular to isolating nucleicacid. As used herein, “isolating nucleic acid” relates to any method inwhich contaminants and impurities are removed from the target nucleicacid and/or in which the concentration of the nucleic acid is enhanced.

The term “nucleic acid” or “nucleic acids” as used herein, in particularrefers to a polymer comprising ribonucleosides and/ordeoxyribonucleosides that are covalently bonded, typically byphosphodiester linkages between subunits, but in some cases byphosphorothioates, methylphosphonates, and the like. Nucleic acidsinclude, but are not limited to all types of DNA and/or RNA, e.g. gDNA;plasmid DNA, circular DNA; circulating DNA; hnRNA; mRNA; noncoding RNA(ncRNA), including but not limited to rRNA, tRNA, lncRNA (long noncoding RNA), lincRNA (long intergenic non coding RNA), miRNA (microRNA), siRNA (small interfering RNA), snoRNA (small nucleolar RNA), snRNA(small nuclear RNA) and stRNA (small temporal RNA), piRNA (piwiinteracting RNA), tiRNA (transcription initiation RNA), PASR (promoterassociated RNA), CUT (cryptic unstable transcripts), extracellular orcirculating RNA; fragmented nucleic acid; nucleic acid obtained fromsubcellular organelles such as mitochondria or chloroplasts; and nucleicacid obtained from microorganisms, parasites, or DNA or RNA viruses thatmay be present in a biological sample. Synthetic nucleic acid sequencesthat may or may not include nucleotide analogues that are added or“spiked” into a biological sample are also within the scope of theinvention. Small RNA or the term small RNA species in particular refersto RNA having a length of less than 500 nt. According to one embodiment,the nucleic acid is DNA.

A sample may comprise more than one type of nucleic acid. Depending onthe intended use, it may be desirous to isolate all types of nucleicacids from a sample ((e.g. DNA and RNA) or only certain types or acertain type of nucleic acid (e.g. only RNA but not DNA or vice versa orDNA and RNA are supposed to be obtained separately). All these variantsare within the scope of the present invention. Suitable methods forisolating either DNA or RNA or both types of nucleic acids in parallelare known in the prior art.

The term “sample” is used herein in a broad sense and is intended toinclude a variety of sources that contain nucleic acids. The sample maybe a biological sample but the term also includes other, e.g. artificialsamples which comprise nucleic acids. Exemplary samples include, but arenot limited to, body fluids in general, whole blood; serum; plasma; redblood cells; white blood cells; buffy coat; swabs, including but notlimited to buccal swabs, throat swabs, vaginal swabs, urethral swabs,cervical swabs, throat swabs, rectal swabs, lesion swabs, abcess swabs,nasopharyngeal swabs, and the like; urine; sputum; saliva; semen;lymphatic fluid; liquor; amniotic fluid; cerebrospinal fluid; peritonealeffusions; pleural effusions; fluid from cysts; synovial fluid; vitreoushumor; aqueous humor; bursa fluid; eye washes; eye aspirates; plasma;serum; pulmonary lavage; lung aspirates; and tissues, including but notlimited to, liver, spleen, kidney, lung, intestine, brain, heart,muscle, pancreas, cell cultures, as well as lysates, extracts, ormaterials obtained from any cells and microorganisms and viruses thatmay be present on or in a sample and the like. Materials obtained fromclinical or forensic settings that contain nucleic acids are also withinthe intended meaning of the term sample. Furthermore, the skilledartisan will appreciate that lysates, extracts, or materials or portionsthereof obtained from any of the above exemplary samples are also withinthe scope of the term sample. Preferably, the sample is a biologicalsample derived from a human, animal, plant, bacteria or fungi. Inparticular, the term “sample” refers to a nucleic acid containing samplewhich also comprises proteins. Preferably, the sample is selected fromthe group consisting of cells, tissue, bacteria, virus and body fluidssuch as for example blood, blood products such as buffy coat, plasma andserum, urine, liquor, sputum, stool, CSF and sperm, epithelial swabs,biopsies, bone marrow samples and tissue samples, preferably organtissue samples such as lung and liver. Preferably, the sample isselected from whole blood and blood products such as buffy coat, serumor plasma.

The nucleic acid may also be an artificially synthesized nucleic acid,for example from a PCR reaction. The sample solution used in the processcould be obtained directly from the biological material, for example bylysis of cells or tissue or by fractionation and/or pre-treatment ofbody liquids. Alternatively, the sample solution could be obtained froma nucleic acid processing method, which may be a method forpurification, synthesis or modification, such as PCR or purificationfrom agarose gel.

In a preferred embodiment, the nucleic acid isolated in the inventivemethod is obtained in pure form, or essentially pure form or it may bedissolved. Preferably, only unavoidable impurities are comprised in thepure nucleic acid or in the dissolved nucleic acid product. Preferably,the purity of the isolated nucleic acid is at least 90%, more preferablyat least 90%, and most preferably more than 99% or 99.5% by weight,referring to the pure nucleic acid or based on all solids in thedissolved nucleic acid product excluding solid additives from theelution solution.

In the inventive process, the nucleic acid preferably is separated fromthe sample solution by binding to magnetic particles. Thus, the nucleicacid must be accessible to the magnetic particles for binding. In apreferred embodiment, the sample solution is an aqueous solution inwhich the nucleic acid is normally dissolved or suspended. In case apreceding starting material does not comprise nucleic acid directlyaccessible for binding, pre-treatment steps are preferably carried out,such as lysis and/or disruption of biological material, such as cells ortissues.

The nucleic acid is separated from the solution by adsorption to themagnetic beads. In the inventive method, binding of the nucleic acid tothe magnetic particles is expected to be mainly adsorptive. In thiscase, the binding usually is non-covalent and reversible. Adsorption isthe adhesion of nucleic acid molecules to the surface of the magneticbeads. In the binding step, conditions preferably are adjusted such thatthe nucleic acid binds to the magnetic particles, typically by addingone or more chaotropic salts, non-chaotropic salts and/or low molecularweight alcohol (“binding-conditions”).

In the inventive method, the binding force between the nucleic acid andthe magnetic particles in step (w) of the inventive process ispreferably only based on adsorption. Preferably, it is not based onother binding mechanisms, such as affinity chromatography or ionexchange chromatography. Affinity chromatography requires a highlyspecific interaction between two specific molecules, such as antigen andantibody, enzyme and substrate, or receptor and ligand, according to thekey-lock-principle. Ion exchange chromatography is based on specificinteractions of target molecules with charged ligands on the substratesurface. More preferably, the method is not based on specificinteractions of nucleic acid with carboxyl groups attached to themagnetic particles.

In a preferred embodiment, the magnetic particles are not modified withligands, especially organic ligands. Preferably, no ligands are attachedto the surface of the particles. Thus, it is preferred that theparticles are not modified with affinity ligands, such as streptavidineor poly-dT. Further, it is preferred that the particles are not modifiedwith ion exchange ligands, such as cation exchange ligands. Morepreferably, they are not modified with carboxyl-groups and/or do notcomprise carboxyl-groups on the surface.

In another embodiment, the magnetic particles are bifunctional. Thismeans that they are capable of adsorbing nucleic acids as a firstfunctionality, but have a second functionality. For example, theadsorptive particles may be modified with affinity ligands, such thatthe second functionality would be affinity binding. This secondfunctionality could be used for binding a substrate to the particles ina different binding step than during (w).

Separation of the magnetic particles with nucleic acid bound theretofrom the sample solution is realized by application of a magnetic field.Typically, a magnetic field is applied outside the sample tube, suchthat magnetic particles are attracted to a defined location andaccumulate there, for example on a side of the sample tube.Alternatively, a magnetic device can be inserted into the sample tube orclose to the sample tube, to which the magnetic particles are adhered.For example, the device could be a magnetic rod. Preferably, in thebinding step magnetic particles accumulate in the form of a pellet orcluster. After accumulating the magnetic particles with the nucleic acidbound thereto, they are separated from the sample solution, therebyremoving the sample solution.

The inventive method comprises at least one washing step (w) whichcomprises at least sub-steps (w1), (w2) and (w3) as outlined above,which are carried out in consecutive order. Preferably, step (w)consists of sub-steps (w1) to (w3).

In sub-step (w1), the magnetic particles with the nucleic acid boundthereto are suspended in the washing solution. In this step, themagnetic particles with the nucleic acid bound thereto are normallydetached from the predetermined location in which they were fixated bythe magnetic field. Preferably, unless the particles are transferredinto another tube the magnetic particles are fixated by the magneticfield such that they maintain contact with the liquid mixture in thetube. Normally, the magnetic field is inactivated before or during step(w1), such that the magnetic particles can be easily suspended in thewashing solution. For example, the magnetic particles may be attached tothe sample tube wall by the magnetic field or to a magnetic devicewithin the tube, whereupon the magnetic field is switched off, removedor relocated and the magnetic particles are suspended in the washingsolution. Preferably, single magnetic particles are distributed in thesolution, such that any cluster or pellet is at least partly, preferablycompletely dispersed. This may be supported by gentle movement.

In a preferred embodiment, suspending the magnetic particles in thewashing solution in step (w1) is supported by mild agitation, preferablyin a vertical direction i.e. in more or less the same direction as thetube axis. In a highly preferred embodiment, the magnetic particles areattracted to a magnet in an elevated position in relation to the bottomof the tube and are dropped into the washing solution within the tube instep (w1) by switching off, removing or relocating the magnet. It wasfound that such mild agitation which in this preferred embodiment may beinduced for example by the particles' movement through the washingsolution to the bottom of the tube for example by gravity or a further,in particular light magnetic force applied below the elevated position,may support release of contaminants from the magnetic beads. Yet, theapplied agitation should be chosen such that no significant release ofthe nucleic acid from the magnetic particles takes place. The gentlemovement may for instance be achieved by a device being dipped into theliquid of the tube and moved up and down, like a magnetic rod or aPickPen® provided e.g. by BioControl. It is also suitable to suspend themagnetic particles by carefully rinsing them from the tube wall or fromany kind of magnetic device that is dipped into the tube liquid with thewashing solution once the magnetic device has been switched off, removedor relocated. Intensive agitation, i.e. in particular induced by amechanical means like any kind of shaking device should be avoided.

Thus, preferably, the magnetic particles with the nucleic acid boundthereto are not subjected to a mechanical force. In a specificallypreferred embodiment the magnetic particles with the nucleic acid boundthereto are not subjected to a mechanical force exerted oscillatingand/or horizontally. Examples for devices exerting oscillating and/orhorizontal mechanical force are the usually known laboratory vortexersor laboratory shakers. Manual shaking is usually also an example forsuch a force. Yet, a mild mechanical force exerted vertically, likegravity, may be applied as well. This may be realized for example by themagnetic beads being attached to the tube wall or to a magnetic devicedipped in the liquid within the tube at an extended position and thenswitching off, removing or relocating the magnetic device. Due to thegravity the magnetic particles sink towards the bottom of the tube,decelerated depending on the viscosity of the liquid in the tube. Inaddition, a magnetic force preferably exerted vertically i.e. havingmore or less the same direction as the tube axis, may be applied. Yet,the strength of the magnetic field should be chosen such that theinduced movement within the washing solution does not significantlyrelease the nucleic acid from the magnetic particles. This may berealized with a magnetic device being positioned outside of the tube,for example at or under its bottom.

In the same way, preferably, the magnetic particles with the nucleicacid bound thereto are suspended in the washing solution withoutapplying a mechanical force. In a specifically preferred embodiment themagnetic particles with the nucleic acid bound thereto are suspended inthe washing solution without applying a mechanical force exertedoscillating and/or horizontally. Yet, a mild mechanical force exertedvertically, like gravity, may be applied when suspending the magneticparticles in the washing solution. In addition, a magnetic forcepreferably exerted vertically i.e. having more or less the samedirection as the tube axis, may be applied.

Thus, the purity of the isolated nucleic acid may be increased furtherby such mild agitation. Further, it is preferred that there is noagitation, i.e. stirring or shaking, in incubation step (w2).

In a preferred embodiment, the magnetic particles with nucleic acidbound thereto in this washing step (w) are not subjected to mechanicalforce, in particular not strong mechanical force, which goes beyond mildagitation. It was found that mechanical forces, and in particular strongmechanical forces, may enhance release of nucleic acid from the magneticparticles. Specifically, the sample should not be stirred or shaken, inparticular not be mechanically stirred or shaken, in the washing step(w).

The washing solution used in step (w1) is an aqueous solution. It ischaracterized by a salt concentration below 100 mM and by the absence ofan organic solvent. Thereby, the washing solution is distinct fromwashing solutions commonly used in such processes with magneticparticles. The washing solution used according to the invention isunique, because in fact conditions are applied which are generallyconsidered as “non-binding” conditions. Thus, washing steps in the priorart are always carried out with washing solutions under “bindingconditions”, which are adjusted for example with a high saltconcentration of 1 M or higher. According to the prior art, the salt isoften a chaotropic salt. Further, typical washing solutions used in theprior art comprise high amounts of organic solvent, especially ethanolor isopropanol. By adding one or more salts at high concentration and/ororganic solvents, it shall be ensured in the processes of the prior artthat the nucleic acid is not detached from the magnetic particles andthereby lost.

As outlined above, for example in the protocol related to theMagAttract® HMW DNA Kit, a final washing step is carried out, in whichthe magnetic particles with nucleic acids attached thereto are rinsedwith pure water. However, the rinsing step is only carried out verybriefly when magnetic particles are adhered in the form of a pellet tothe sample tube wall. Care should be taken that the pellet is notdisrupted or mechanically disturbed when the water is added at adifferent position in the tube. When proceeding accordingly, the rinsingwater has no access to the interior of the magnetic particle pellet,such that the high salt and ethanol environment is basically maintainedwithin the pellet. The method of the present invention is strikinglydifferent because the magnetic particles with the nucleic acids boundthereto are suspended in the low-salt washing solution. In the priorart, it had been assumed that such a treatment would not be applicablefor washing, because it should cause elution of nucleic acids from theparticles. Surprisingly, it has been found according to the presentinvention that washing may be carried out in a suspension under such“non-binding” conditions, with a great increase in product purity, butwithout significant loss of nucleic acid. This is highly advantageous,because an efficient removal of salt, ethanol and/or other contaminantsfrom the target nucleic acid can be achieved without significantreduction of the nucleic acid yield. Without being bound to theory, itis assumed that an intermediate state is formed, in which forces betweenthe magnetic particles and nucleic acid molecules temporarily preventrelease of the nucleic acid molecules from the particles. In contrast,in an equilibrium state, which is not reached in the inventive process,a significant loss of nucleic acid would be obtained. The inventiveprocess is also highly advantageous, because a drying step forevaporating organic solvents, such as ethanol, although still possible,is not necessary any more. The inventive process correspondingly maysave considerable time, because drying steps for removal of organicsolvents, if carried out thoroughly, are relatively time-consuming.

As noted above, the total salt concentration of the washing solution ispreferable below 100 mM. Such a concentration is in general insufficientfor stable binding of nucleic acids to magnetic particles by adsorption.In contrast, low salt concentrations typically destabilize thenon-covalent interactions between the magnetic particle surfaces and thepolyanionic nucleic acid. In a preferred embodiment, the washingsolution has a total salt concentration of below 50 mM, more preferablyof below 25 mM or of below 10 mM. In another preferred embodiment, thewashing solution does not comprise any salt. This means that salt hasnot been added to the washing solution. Thus, only unavoidably saltimpurities like the ones already incorporated within the magneticparticle/nuclei acid complex may be comprised. Preferably, the washingsolution is water, more preferably distilled water. In principle,including salt into the washing solution in a concentration of up to 100mM is not required for stabilizing the magnetic particle/nucleic acidcomplex in washing step (w). However, a low amount of salt may bedesired in the final product, for example in the form of a buffersubstance for stabilizing the product or facilitating further use. Thus,the salt could be a buffer substance, such as TRIS(Tris(hydroxymethyl)aminomethan). The pH of the washing solution may bebetween 7 and 10, especially between 7.5 and 9.

In a preferred embodiment, the conditions in the liquid phase of thesample (the suspension) in step (w1), after addition of the washingsolution, are essentially the same as in the washing solution used instep (w1), at least with respect to salt concentration and absence of anorganic solvent. Therefore, it is preferred that before addition of thewashing solution in step (w1), the sample does not comprise significantamounts of residual salt or organic solvent, which would significantlyincrease the salt concentration or level of organic solvent in theliquid phase. Therefore, it is preferred that the total saltconcentration in the liquid phase, in the suspension of step (w1), afteraddition of the washing solution, is below 100 mM, more preferably ofbelow 25 mM or of below 10 mM. Further, it is preferred that suspensiondoes not comprise organic solvent, except for unavoidable impurities,for example below 2 wt.%. In a preferred embodiment, the saltconcentration in the solution in the process step which is carried outdirectly before step (w) (or if step (w) is repeated, before the laststep (w)) is not more than 2 M, preferably not more than 1 M.

If desired, the washing solution may comprise additives different fromsalts and organic solvents, such as compounds for stabilizing nucleicacids, such as proteases or complexing agents like EDTA. In a preferredembodiment, the washing solution consists of water and optionally saltat a concentration of below 100 mM, and optionally such additives.

The washing solution in washing step (w) preferably does not comprise anorganic solvent. Thus, in a preferred embodiment it does not compriselow molecular weight alcohols having 1 to 5 carbon atoms, such asethanol or isopropanol. Thus, the washing solution is distinct fromtypical conventional washing solutions used in such processes whichcomprise ethanol or isopropanol. In a specific embodiment, the washingsolution does not comprise an organic compound at all. Preferably, thewashing solution does not comprise a detergent.

It is highly preferred that washing step (w) is the final washing stepin the inventive method. In a specifically preferred embodiment, thewashing step (w) is directly followed by the final elution of thenucleic acid from the magnetic particles. In general, it is preferredthat the washing solution used in step (w) is identical to the elutionsolution. Yet, it is also possible to release the nucleic acid from themagnetic particles after the washing step (w) not in a separate elutionstep but in the subsequent downstream procedure.

In a preferred embodiment of the invention, washing step (w), i.e. thecombination of sub-steps (w1) to (w3), is carried out in 5 minutes orless, preferably 2 minutes or less. This means that it should be carriedout in the defined time range. In other words, the time in which themagnetic particles with nucleic acid adsorbed thereto are in contactwith the washing solution should be in the defined time range.

In sub-step (w2), the magnetic particles with the nucleic acid boundthereto are incubated in the washing solution. The incubation time isadapted in order to remove as much contaminants as possible whilstavoiding significant loss of nucleic acid. The shorter the incubationtime, the more contaminants may remain in the product. Thus, it may bepreferable that the incubation time in step (w2), and/or the overalltime for step (w), which is mostly determined by the incubation time, isat least 3, at least 5, at least 10, at least 20 or at least 30 seconds,or at least 1 minute. The higher the incubation time, the higher will bethe risk of loss of nucleic acid. This applies all the more the shorterthe nucleic acid is. Thus, it may be preferable that the incubation timeand/or time of step (w) is 20 minutes or less, 10 minutes or less, 5minutes or less, 2 minutes or less, 1 minute or less, 30 seconds orless, 20 seconds or less, 10 seconds or less, preferably 5 seconds. Forexample, the time could be between 3 seconds and 20 minutes, preferablybetween 5 seconds and 10 minutes, between 10 seconds and 5 minutes,between 20 seconds and 2 minutes or between 30 seconds and 1 minute. Insuch time spans, a relatively good balance between high loss ofcontaminants and high nucleic acid yield can be achieved.

In sub-step (w3), the magnetic particles with the nucleic acid boundthereto are separated from the washing solution. Preferably, themagnetic particles with the nucleic acid bound thereto are separatedfrom the washing solution by application of a magnetic field.Preferably, the means of removing magnetic particles from the solutionare the same throughout the entire process.

In the inventive method, the nucleic acid to be isolated is notspecifically limited. The DNA could be genomic DNA (gDNA), plasmid DNA(pDNA), DNA fragments of any length, e.g. produced from restrictionenzyme digestion, amplified DNA of any length produced by anamplification reaction such as the polymerase chain reaction (PCR) ornucleic acid sequence-based amplification (NASBA), DNA resulting from abioprocess sample as obtained during the production of biotechnologicalcompounds like biopharmaceuticals, or the like, wherein the DNA may bedouble-stranded or single-stranded. The term RNA as used in the presentinvention comprises but is not limited to total RNA, mRNA, rRNA or tRNA.Other suitable nucleic acids have been described above.

According to the invention, efficient removal of contaminants, such asnon-target biomolecules, salts and organic solvents, is possible withnucleic acid of all chain lengths. For example, the DNA could have achain length from 10 bp to 100 kbp, preferably from 500 bp to 50 kbp, orfrom 1 kbp to 20 kbp, the RNA having the corresponding chain lengths inbases or nucleotides.

In a preferred embodiment of the invention, the nucleic acid has a chainlength of 2 kbp or more, preferably 5 kbp or more, or more preferably 10kbp or more or the corresponding amount of bases or nucleotides,respectively. In a preferred embodiment, the nucleic acid may be genomicDNA or plasmid DNA. When the nucleic acid has a higher chain length like10 kbp or above, more preferred 20 kbp or above most preferred 50 kbp orabove, loss of nucleic acid in the washing step is considerednegligible. Then, the washing step (w) may take a relatively longoverall time in the range of minutes, such as up to 10 minutes. Yet, inparticular for reasons of convenience a shorter overall washing time asindicated above may be preferred.

Some reduction of the yield may be observed with nucleic acids, inparticular DNA having a relatively short chain length. However, loss ofnucleic acid can then be avoided by decreasing the time of the washingstep (w) and/or the incubation time in step (w2), for example to 2minutes or less, 1 minute or less, 30 seconds or less, 20 seconds orless, 10 seconds or less, 5 seconds or less or 3 seconds. In principle,the shorter the DNA is, the less time washing step (w) should take.Under those circumstances the loss in nucleic acid may be in similarorder of magnitude in the present method of the invention as in therinsing procedure of the state of the art but with the additionaladvantage that more contaminations are removed resulting in nucleic acidof higher purity than in the state of the art.

In a preferred embodiment of the invention, the nucleic acid has a chainlength of 30 kbp or less, 20 kbp or less, or 10 kbp or less, or acorresponding number of bases or nucleotides, wherein washing step (w)is carried out in 2 minutes or less, 1 minute or less, 30 seconds orless, 20 seconds or less, 10 seconds or less or preferably 5 seconds.Preferably the nucleic acid is DNA. In other preferred embodiments, thenucleic acid has a chain length of 5 kbp or less, or 3 kbp or less, 2kbp or less, 1 kbp or less, more preferred at least 500 bp, or acorresponding number of bases or nucleotides, wherein the washing stepis carried out in 1 minute or less, or 30 seconds or less, 20 seconds orless, 10 seconds or less, 5 seconds or less or 3 seconds. Preferably thenucleic acid is DNA.

Washing step (w) may be repeated as often as desired, for example may berepeated once, twice, three times or even more often. However, it wasfound that a very efficient removal of impurities is possible in asimple process with only a single washing step. Thus, it is highlypreferred to carry out washing step (w) only once.

In a preferred embodiment, washing step (w) is carried out at atemperature of 30° C. or less, preferable between 0° C. and 30° C. Ifthe temperature in washing step (w) is too high, nucleic acid may bereleased from the magnetic particles and get lost. In a preferredembodiment, it is carried out at room temperature, for example at fromabout 15° C. to 25° C. However, the sample may also be cooled, forexample to 15° C. or less, most preferably 5° C. or less.

In a preferred embodiment, the method comprises additional washingsteps, which are different from washing step (w). Preferably, suchadditional washing steps are carried out before washing step (w). Suchadditional washing steps may be carried out with conventional washbuffers or solutions comprising ethanol as described in the prior art.With such additional washing steps, a pre-purification can be achieved,such that levels of salt, alcohol and/or other contaminants aredepleted. In a highly preferred embodiment, washing step (w) is the lastwashing step before an optional elution step.

In a preferred embodiment, before washing step (w) the magneticparticles with the nucleic acid adsorbed thereto are subjected to atleast one pre-washing step (p) in which the washing solution is adaptedto nucleic acid binding conditions. This means that in the pre-washingstep (p), the salt concentration is relatively high and/or organicsolvent is comprised. Such washing steps under binding conditions arecommonly used in known methods to remove contaminants from the samplesolution. Also in the inventive process, it may be advantageous toinclude such pre-washing steps (p) to remove contaminants, such asproteins or other cell components, or undesired low molecular weightcompounds, from the sample solution.

In a preferred embodiment of the invention, before washing step (w), themagnetic particles with the nucleic acid adsorbed thereto are washed inat least one pre-washing step (p) with a washing solution, which is anaqueous solution having a total salt concentration above 500 mM and/orcomprises an organic solvent. More specifically, in the prewashing step(p), the total salt concentration of the prewashing step (p) washingsolution could be above 1 M or even above 2 M. In prewashing step (p),the organic solvent is preferably ethanol or isopropanol. Specifically,in the prewashing step (p), the washing solution may comprise a loweralcohol, such as ethanol or isopropanol, at a concentration of at least10%, or at least 50% by weight, or pure alcohol.

In an embodiment of the invention, an additional washing step (p) iscarried out, in which the magnetic particles with the bound nucleicacid, when fixated by the magnetic field, are rinsed with water, or witha low salt buffer as used in washing step (w), such that the nucleicacid remains bound to the particles. However, since washing step (w) ismore efficient than such a rinsing step, it is preferred that such arinsing step is not comprised.

In a preferred embodiment of the invention, after washing step (w) thenucleic acid is eluted from the magnetic particles with elutionsolution. Preferably, if elution is desired, the elution step is carriedout directly after washing step (w). In other words, no otherintermediate treatment steps, and especially washing steps, are carriedout after washing step (w). The elution solution is preferably purewater, typically distilled water, or an elution buffer having a low saltconcentration. Typically, elution buffer has a salt concentration of 25mM or less, wherein the salt is generally a buffer salt, such as 10 mMTRIS. The pH of the elution solution and/or buffer is typically between7 and 10, especially between 7.5 and 9. After elution, the nucleic acidpreferably remains in the resulting elution solution while magneticparticles are removed from the elution solution by external force, suchas centrifugation or a magnetic field. Yet, it is also suitable to elutethe nucleic acid by the conditions applied in a subsequent downstreamtreatment and not in a separate elution step preceding the downstreamtreatment.

After the washing step (w), the target nucleic acid can be eluted formthe magnetic beads with an elution solution. This elution step should beclearly distinguished from washing step (w), although the elutionsolution may be identical to the washing solution used in washing step(w). In the washing step (w), the nucleic acid essentially shall remainbound to the magnetic particles, whereas in the elution step theconditions are adjusted such that the nucleic acid is supposed to beeluted.

Preferably, elution is supported by mechanical force, extendedincubation time and/or increased temperature. In a preferred embodiment,the elution of the nucleic acid from the magnetic particles is enhancedby applying a mechanical force. Specifically, elution may be acceleratedby intensive stirring or shaking, especially at high speed, such asvortexing. The elution can be supported by incubating the magneticparticles with the nucleic acid adsorbed thereto in the elution solutionfor an extended time period, such as more than 5 minutes, more than 10minutes or more than 60 minutes. Increasing the temperature may supportthe release of the nucleic acid from the magnetic particles. Thus,elution may be carried out at a temperature of 20° C. or higher, such asroom temperature, or above 30° C.

The term ‘magnetic particles’ as used herein comprises magneticallyresponsive particles which are able to reversibly bind nucleic acids inthe terms of the present invention, e.g. by ionic interaction or thelike. Such particles are well known to a person skilled in the art. Asused herein, the term ‘magnetic’ encompasses magnetic materials, such asferromagnetic, ferrimagnetic, paramagnetic or superparamagneticmaterials. These magnetic materials are part of the magnetic particlesas described above and may be included in the particles by any suitablemethod.

In a preferred embodiment of the invention, the magnetic particlescomprise silica. The silica could be in the form of silica gel,siliceous oxide, solid silica such as glass or diatomaceous earth, or amixture of two or more of the above. The silica should be present atleast in part on the surface of the beads. For efficient binding, it ispreferred that the silica is present on the entire surface of theparticles.

The magnetic particles may be silica coated magnetic particles. The term‘silica coated magnetic particles’ refers to magnetic particles coatedwith silica. These particles are well known to the artisan. Any kind ofsilica coated magnetic particles is in principal suitable for the methodof the present invention. Yet, preferably the magnetic particles are noanion exchange particles.

The silica gel is preferably chromatography grade silica gel, asubstance which is commercially available from a number of differentsources. Silica gel is most commonly prepared by acidifying a solutioncontaining silicate, e.g. sodium silicate, to a pH of less than 10 or 11and then allowing the acidified solution to gel (e.g. silica preparationdiscussion in Kurt-Othmer Encyclopedia of Chemical Technology, Vol. 6,4th ed., Mary Howe-Grant, ed., John Wiley & Sons, pub., 1993, pp.773-775).

The glass particles are preferably particles of crystalline silica(e.g., a-quartz, vitreous silica), even though crystalline silica is notformally ‘glass’ because it is not amorphous, or particles of glass madeprimarily of silica. Glass particles are also well known to the skilledperson.

Siliceous-oxide coated magnetic particles are the most preferred form ofsilica magnetic particles used in the present invention. Siliceous-oxidecoated magnetic particles are comprised of siliceous oxide coating acore comprising at least one particle of ferrimagnetic, ferromagnetic,superparamagnetic or paramagnetic material. The siliceous-oxide coatedmagnetic particles used in the present invention also have an adsorptivesurface of hydrous siliceous oxide. The target nucleic acid, such as DNAor RNA, adheres to the adsorptive surface of the particles while othermaterial from the source of the nucleic acid, particularly deleteriouscontaminants such as nucleases or the like, do not adhere to or co-elutefrom the siliceous-oxide coated magnetic particles together with thenucleic acid. Siliceous-oxide coated magnetic particles are well knownto the artisan and are commercially available (e.g. MagAttract® magneticparticles, QIAGEN, Hilden, Germany).

The magnetic particles have the capacity to form a complex with thenucleic acid in the aqueous solution by reversibly binding the nucleicacid, e.g. by ionic interaction or the like. The present invention maybe performed using any silica coated magnetic particles possessing theproperty of forming a complex with the nucleic acid. Even morepreferably, the method of the present invention is performed using anyform of siliceous-oxide coated magnetic particles, e.g. MagAttract®magnetic beads (QIAGEN, Hilden, Germany). The silica coated magneticparticles used in the methods of the present invention may be any one ofa number of different sizes. Smaller silica magnetic particles providemore surface area per weight unit for adsorption, but smaller particlesare limited in the amount of magnetic material which can be incorporatedinto such particles compared to larger particles.

The term ‘salt’ as used herein preferably refers to chaotropic andnon-chaotropic salts. Typically, the sample solution and/or washingbuffer for pre-washing steps (p) comprise a chaotropic salt and/or a nonchaotropic salt. It was found that such salts can be efficiently removedin the inventive method by washing step (w).

In a preferred embodiment chaotropic salts are salts of chaotropic ionsaccording to the ‘Hoffmeister-Reihe’. Such salts are highly soluble inaqueous solutions. The chaotropic ions provided by such salts, atsufficiently high concentration in aqueous solutions of proteins ornucleic acids, cause proteins to unfold, nucleic acids to lose secondarystructure or, in the case of double-stranded nucleic acids, melt (i.e.strand-separation). It is assumed that chaotropic ions show theseeffects because they disrupt hydrogen-bonding networks that exist inaqueous solutions and thereby make denatured proteins and denaturednucleic acids thermodynamically more stable than their correctly foldedor structured counterparts. The chaotropic salt in the sample solutionmay be selected from the group of guanidinium isothiocyanate,guanidinium thiocyanate, guanidinium hydrochloride, sodium iodide,potassium iodide, lithium chloride, sodium perchlorate, sodiumtrichloroacetate or a mixture thereof. In a preferred embodiment of theinvention, the non-chaotropic salt in the sample solution is selectedfrom the group of sodium chloride, potassium chloride, ammoniumchloride, calcium chloride, magnesium chloride or a mixture thereof.

The salt concentration in the sample solution of the present inventionis preferably in a range of from 0.5 M and 10 M. With any salt used inthe invention, it is desirable that the concentration of the salt, inany of the solutions in which the salt is employed in performing themethod of the invention, remains below the solubility of the salt in thesolution under all of the conditions to which the solution is subjectedin performing the method of the invention. The concentration of the saltin the mixture must be sufficiently high to cause the biopolymer toadhere to the silica magnetic particles in the mixture, but not so highas to substantially denature or to degrade the nucleic acid, or to causethe nucleic acid to precipitate out of the aqueous solution. Proteinsand large molecules of double-stranded DNA, such as chromosomal DNA,usually are stable at chaotropic salt concentrations between 0.5 M and 2M, but are known to precipitate out of solution at chaotropic saltconcentrations above about 2 M (e.g. U.S. Pat. No. 5,346,994, column 2,lines 56-63). Contrastingly, RNA and smaller molecules of DNA such asplasmid DNA, restriction fragments or PCR fragments of chromosomal DNA,or single-stranded DNA usually remain undegraded and in solution atchaotropic salt concentrations between 2 M and 5 M, which is well knownto those skilled in the art. Thus, the salt concentration in a methodaccording to the invention is dependent on the area of application andis apparent to those skilled in the art or is readily determinable.

If desired, the sample solution may comprise at least one additive forenhancing the binding to the magnetic particles. For example, theadditive could be a non-ionic substance which is strongly hydratable andis selected from the group of ethylene glycol, tetraethylene glycol,polyalkylene glycol, cyclodextrin, carrageenan, dextran, dextransulfate, xanthan, cellulose, hydroxypropyl cellulose, amylose,2-Hydroxypropyl β-cyclodextrin, Agar Agar, or is a mixture thereof.Polyalkylene glycol is preferable but not restricted to polyethyleneglycol, polypropylene glycol or a mixture thereof. In a preferredembodiment, the additive is polyethylene glycol. These additives areable to inhibit substantially the clustering of the magnetic particlesin an aqueous solution at relatively low concentrations which allows fora trouble-free automated process, e.g. no clogging of pipette tips dueto clustered magnetic particles occurs. Further information regardingthe selection and use of such additives is provided in WO2005/021748 A1.

In a preferred embodiment of the invention, the method comprises thesteps of

-   -   (a) adding magnetic particles to a sample solution comprising        the nucleic acid,    -   (b) incubating the sample solution of step (a) to adsorb the        nucleic acid to the magnetic particles,    -   (c) applying a magnetic field to the sample solution to fixate        the magnetic particles with the nucleic acid adsorbed thereto,    -   (d) removing the sample solution,    -   (e) washing the magnetic particles with a washing solution in a        washing step (w) as defined above, and    -   (f) eluting the nucleic acid from said magnetic particles with        an elution solution.

Preferably, steps (a) to (f) are carried out in consecutive order.Further steps may be included in the process, such as a pre-treatment ofthe original sample before step (a) for example in order to render thenucleic acid accessible for binding, like a lysis procedure releasingthe nucleic acid, and/or in order to adjust the binding conditions.Before washing step (e), pre-washing steps (p) may be carried out inorder to remove contaminants from the nucleic acid and magneticparticles. Washing step (e), i.e. washing step (w) under non-bindingconditions, may be repeated until salt and organic solvent is removed asdesired. In a specific embodiment, the method may basically consist ofsteps (a) to (f) and (p), each of which may be repeated if desired. Inprinciple, methods with steps (a) to (d) and (f) are known in the art,and thus these process steps can be carried out as described in theprior art.

Preferably, the inventive process, or at least the steps of providing asample solution, binding and washing, and optionally also elution,is/are carried out in a single tube. Typically, after the magneticparticles were treated with a solution and removed from the solution,the solution is also removed from the tube before the next step iscarried out.

In a preferred embodiment of the invention, the method is an automatedprocess. Since the inventive process is relatively simple to realize, itis especially suited for automation, for example with robots or workstations as commonly used in the prior art. Various methods of automatedseparation of magnetic particles are known from the art and all shouldbe suitable for use in the present invention. As an example, a magneticor magnetizable device can be inserted into the medium containing themagnetic particles, the magnetic particles are bound to the magnetic ormagnetizable device, and the magnetic or magnetizable device is removedagain. In a second embodiment of the inventive method the separation ofmedium and the magnetic particles, both aspirated into a pipette tip, isfacilitated by a magnetic or magnetizable device which is brought intospatial proximity to the pipette tip. The magnetic particles are keptback in the pipette tip for example at the wall or the bottom of the tipwhen the medium is removed from the pipette tip. Automation of these twomethods generally requires special technical means, e.g. a robotconstructed especially for one of those methods. Yet, the methods mayalso be conducted manually. A more general principal in removingmagnetic particles is to bring a magnetic or magnetizable device intospatial proximity to the container containing the medium and themagnetic particles. The magnetic particles bind to the container walland the medium can be removed without carry-over of magnetic particles.

Subject of the invention is also the use of an aqueous solution, whichhas a total salt concentration below 100 mM and does not comprise anorganic solvent, as a washing solution for washing magnetic particleswith nucleic acid adsorbed thereto in a method for isolating nucleicacid from a sample solution, wherein the magnetic particles with thenucleic acid adsorbed thereto are suspended in the washing solution.Preferably, the use is in a method as described above, and/or comprisesspecific steps or features as described above for the inventive method.

The nucleic acid using the method of the present invention may beobtained from biological material, such as eukaryotic or prokaryoticcells in culture or from cells taken or obtained from tissues, tumorcells, exosomes, multicellular organisms including animals and plants;body fluids such as blood, lymph, urine, feces, or semen; food stuffs;cosmetics; or any other source of cells or they may be extracellular.Further suitable sample materials have been described above. Somebiopolymers, such as certain species of DNA or RNA are isolatedaccording to the present method from the DNA or RNA of organelles,viruses, phages, plasmids, viroids or the like that infect cells. Thecells could be bacterial cells, such as E. coli cells. Cells will belysed and the lysate usually processed in various ways familiar to thoseskilled in the art to obtain an aqueous solution of DNA or RNA, to whichthe separation or isolation methods of the invention are applied. TheDNA or RNA, in such a solution, will typically be found with othercomponents, such as proteins, RNA (in the case of DNA separation), DNA(in the case of RNA separation), or other types of components. Furthersuitable biopolymers have been described above.

Before contacting the magnetic particles with the nucleic acid, it maybe necessary to disrupt biological material in which the nucleic acid iscontained. Regardless of the nature of the source of the nucleic acid,the nucleic acid to be isolated with the method of the present inventionis provided in an aqueous solution comprising the nucleic acid andmolecule different from the nucleic acids, e.g. cell debris,polypeptides, etc. When the nucleic acid material to be isolated usingthe methods of the present invention is contained within a cell, ortissue comprising cells, it is preferably first processed by lysing ordisrupting the cell or tissue to produce a lysate (herein referred to as‘crude lysate’), and more preferably additionally processed by clearingthe lysate of cellular debris (e.g. by centrifugation or vacuumfiltration). Any one of a number of different known methods for lysingor disrupting cells to release nucleic acid materials contained thereinare suitable for use in producing an aqueous solution from cells for usein the present invention. The method chosen to release the nucleic acidmaterial from a cell will depend upon the nature of the cell containingthe material. For example, in order to cause a cell with a relativelyhard cell wall, such as a fungus cell or a plant cell, to release thenucleic acid material contained therein one may need to use harshtreatments such as potent proteases and mechanical shearing with aparticle mill or a homogenizer, or disruption with sound waves using asonicator. Contrastingly, nucleic acid material can be readily releasedfrom cells with lipid bilayer membranes such as E. coli bacteria oranimal blood cells merely by suspending such cells in an aqueoussolution and adding a detergent to the solution. Once the nucleic acidmaterial is released from the cells lysed or disrupted as describedabove, cellular debris likely to interfere with the adhesion of thenucleic acid material to magnetic particles can be removed using anumber of different techniques known from the art or combination ofthese techniques. The crude lysate is preferably centrifuged to removeparticulate cell debris. Optionally, the supernatant is subsequentlyfurther processed by adding a second solution to the supernatant whichcauses a precipitation of other cell constituents, e.g. polypeptides,and then removing the precipitate from the resulting solution bycentrifugation. In a preferred embodiment, the lysate thus obtained, orthe supernatant, is used as the sample solution in the inventiveprocess.

The nucleic acid may also be obtained an artificial source. The nucleicacid material can be the product of an amplification reaction, such asamplified DNA produced by the polymerase chain reaction (PCR) or nucleicacid sequence-based amplification (NASBA), or the like. The nucleic acidmaterial can also be in the form of fragments of any length, e.g.produced from restriction enzyme digestion. The aqueous solutionaccording to the invention may also be an aqueous solution comprisingmelted or enzymatically digested electrophoresis gel and nucleic acidmaterial.

The nucleic acid isolated by the method of the present invention issuitable, without further isolation or purification, for analysis orfurther processing by molecular biological procedures, isolated nucleicacids can be analysed by, for example, sequencing, restriction analysis,or nucleic acid probe hybridization. Thus, the methods of the inventioncan be applied as part of methods, based on analysis of DNA or RNA, for,among other things, genotyping, diagnosing diseases, identifyingpathogens, testing foods, cosmetics, blood or blood products, or otherproducts for contamination by pathogens, forensic testing, paternitytesting, and sex identification of fetuses or embryos or otherpreferably non-invasive prenatal testing (NIPT).

DNA or RNA isolated by the method of the present invention may beprocessed by any of various exonucleases and endonucleases that catalysereactions with DNA or RNA, respectively, and, in the case of DNA, can bedigested with restriction enzymes, which cut restriction sites presentin the DNA. Restriction fragments from the eluted DNA can be ligatedinto vectors and transformed into suitable hosts for cloning orexpression. Segments of the eluted DNA or RNA can be amplified by any ofthe various methods known in the art for amplifying target nucleic acidsegments. If eluted DNA is a plasmid or another type of autonomouslyreplicating DNA, it can be transformed into a suitable host for cloningor for expression of genes on the DNA which are capable of beingexpressed in the transformed host.

The inventive method solves the problem underlying the invention. Anefficient method for isolation of nucleic acids is provided, whichovercomes the drawbacks of known methods. Nucleic acid can be isolatedin pure form and at high yield in a relatively simple and convenientprocess. Impurities, such as salts, organic solvents and contaminantsfrom the sample solution are efficiently removed. Nucleic acids ofdifferent chain lengths can be isolated at high yield when adapting theincubation time in the washing step (w). The process does not require atime-consuming drying step for evaporation of organic solvents. Thechemicals and components for washing step (w) are simple, because thewashing solution can be water or low salt aqueous solution, and may evenbe identical to the elution solution.

Further aspects of the invention are shown in the figures.

FIG. 1 shows the results of example 3 in graphical form. Impurities inthe eluents were analysed spectrometrically in the range of 220 to 350nm. Dotted lines relate to two probes with DNA isolated by the triplewash method (comparative) according to example 1. Continuous linesrelate to two probes with DNA isolated by a water-drop method accordingto the invention of example 2.

FIG. 2 shows the results of example 4 in graphical form. Impurities inthe eluents were analysed spectrometrically in the range of 220 to 350nm. Dotted lines relate to three probes with DNA isolated by the triplewash method (comparative). Continuous lines relate to a probe with DNAisolated by a water-drop method according to the invention.

FIG. 3 shows an agarose gel of example 5 with genomic DNA isolated fromE. coli according to example 2. DNA was analyzed from the eluate (lanea) and from the water used in the water-drop washing step (lane b).

FIG. 4 shows an agarose gel of example 6 with plasmid pCMVβ isolatedfrom E. coli according to example 2. DNA was analyzed from the eluate(“elution”) and from the water used in the water-drop washing step(“water-drop”).

FIG. 5 shows an agarose gel of example 6 with plasmid pCMVβ isolatedfrom E. coli according to example 2. DNA was analyzed from the eluate(“B: Eluate”) and from the water used in the water-drop washing step(“A: Water from water-drop”) after different incubation times of 0, 6,12, 18 and 24 h. Further results are shown in section C from secondelution step subsequent to the elution step in B., wherein mechanicalforce and moderate heat was applied to suspensions in the second elutionstep.

FIG. 6 shows an agarose gel of example 8 with plasmid pCMVβ and pUC21isolated from E. coli according to examples 1 and 2. DNA was analyzedfrom the eluate (“elution”) and from the water used in the water-dropwashing step (“drop”) or rinsing step (“rinse”), respectively.

EXAMPLES Examples 1 and 2: Purification of DNA

DNA from bacterial cells was isolated according to the method of theinvention (example 2) and to a prior art method (example 1). Conditionsand working solutions are listed in table 1 below. Lysis of E. coli orwhole-blood cells was performed according to the standard protocol witha kit available under the trademark QIAamp® (Qiagen AG, Hilden, Germany;cat no. 51304). DNA was purified with magnetic particles with theMagAttract® kit according to the standard protocol (Qiagen AG, Hilden,Germany; cat. no 67563) using a magnetic bead extraction platform(trademark Gilson Extractman®; Gilson Inc., Middleton, US/SalusDiscovery). In the standard process of example 1, DNA was bound tomagnetic silica particles from a buffer comprising a high concentrationof chaotropic salt to establish binding conditions. Magnetic particleswere separated from the sample solution by application of a magneticfield. Specifically, the magnetic particles were pulled out of thesolution, followed by removal of sample solution. In lysis step 1(table), the beads were only taken up and not suspended in the lysateagain. The magnetic particles with DNA bound thereto were washedaccording to the protocol with various standard wash buffers by releaseof the particles into a washing buffer in a sample well, washing andre-attracting/collecting the magnetic particles with the bound DNA.Release was carried out by switching off, removing or relocating themagnetic field, such that the magnetic beads with nucleic acid boundthereto dropped into the solution below. In most washing steps, thiscylce of binding the particles and “dropping” them was repeated in orderto impose some mild mechanical force onto the particles. The number of“drops” of the magnetic beads in each step is also shown in the table.

In comparative example 1, before elution, the magnetic particles withDNA bound thereto, which were fixed to the sample tube by a magneticforce, were carefully rinsed with distilled water in step (5a). Nofurther mechanical force, such as shaking or stirring, was applied inthis washing step. In the following, this step is referred to as a“water-rinse”-step.

In the inventive example, the final water-rinse step was replaced by afinal washing step (5b) in which the magnetic particles with DNA boundthereto were suspended in distilled water. The magnetic particles werereleased by removing the magnetic field and dropping the magneticparticles into the washing solution, followed by incubation for 5-10seconds. No further mechanical force, such as shaking or stirring, wasapplied in this washing step. Subsequently, the magnetic particles withnucleic acids bound thereto were separated from the washing solution byre-initiating the magnetic force. In the following, such a washing step(w) is referred to as a “water-drop”-step, because the magneticparticles are dropped into the water for washing.

Elution was carried out by repeatedly dropping the beads into theelution solution in order to improve release of nucleic acid. Elutionwas supported by magnetic stirring for 3 minutes.

TABLE 1 Experimental conditions with buffer used and number of washingsteps. In each step, the magnetic particles were separated from thesolution and the solution was removed. The number of “drops”, i.e.repeated release of magnetic particles into the same solution, is alsoindicated. Ex. 1 (comp): Ex. 2 Amount [number [number Step BufferComposition [μl] of drops/rinses] of drops] 1 Lysate crude cell lysate400 3^(d) 3 guanidinium HCl ethanol 2 wash 1.5% Tween 20 250 3^(d) 3 MW17.5M guanidinium HCl 370 mM LiCl 3 wash 19 mM TRIS 120 3^(d) 3 PE 80%ethanol 4 wash 19 mM TRIS 120 3^(d) 3 PE 80% ethanol  5a “water- 19 mMTRIS 120 1 ^(r) — rinse” 80% ethanol  5b “water-drop” distilled water120 — 1 6 elution AE 10 mM TRIS 120 10^(d)  10 0.5 mM EDTA ^(d)= drops,^(r) = rinses

Example 3: Purity of Genomic DNA from E. coli

Impurities in E. coli genomic DNA prepared according to examples 1 and 2above was determined spectrometrically. In order to wash offcontaminants from the magnetic particles, elution was carried out bysubjecting magnetic particles for three minutes to a magnetic stirrer.Impurities in the eluents were analyzed with a spectrometer in the rangeof 220 nm to 350 nm, wherein mostly salt carry-over is determined. Theresults are shown in FIG. 1. Dotted lines show values detected for twoprobes with DNA isolated according to a water-rinse protocol of example1 (comparative). Continuous lines show values detected for two probeswith DNA isolated according to a water-rinse protocol of example 2. Ahigh OD value in the range between 220 and 245 nm is generallyindicative of salt and other contaminants. The results demonstrate thatthe inventive process, with washing of magnetic particles with bound DNAin distilled water under non-binding conditions, removes far morecontaminants than the comparative method with a water-rinse step of theprior art.

Example 4: Purity of DNA from Blood

DNA from human whole-blood was prepared according to the protocol ofinventive example 2 and modified comparative example 1. In the processof example 1 described above, water-rinse step 5a (see table) wasreplaced by a third washing step with PE buffer. As in first and secondPE buffer washing steps 3, 4, also in the third PE puffer washing stepthe magnetic particles were dropped three times into the solution. Thepurities of the isolated DNA products were compared. The aim was todetermine if the additional washing step in the comparative process,with 3 drops into ethanol buffer PE, may remove impurities moreefficiently than the inventive process with only one single drop of theparticles into water.

The results are shown in FIG. 2. Dotted lines relate to DNA isolated bythe comparative triple wash method. Continuous lines relate to DNA ofexample 2 isolated by the water-drop method. The results demonstratethat the inventive process can remove significantly more contaminantsfrom the sample than a conventional process. Each drop of the magneticparticles into washing buffer is associated with a gentle mechanicalforce that supports washing the magnetic particle-bound DNA free ofcontaminants. Following that logic, a triple sample drop into a washbuffer should be superior to a single drop in removing salts and othercontaminants from the sample. Thus, it was unexpected that a single-dropinto water cleaned up the DNA-sample similarly well or better than atriple washing step, wherein the particles were dropped into standardwashing buffer.

Example 5: Yield of Genomic DNA from E. coli

The “water-drop” step in the inventive process was carried out withwater, which usually imposes elution conditions to DNA bound to magneticparticles. Therefore, it was determined if a loss of DNA occurs in themethod described in the examples above. Genomic DNA (gDNA; about 20 kbplength) was prepared from E. coli in the method of example 2 above. DNAfrom the eluate and from the remaining water used in the water-dropwashing step was analyzed by agarose gel electrophoresis. The resultsare shown in FIG. 3. A strong gDNA band is visible for the product inthe eluate (lane a), whereas DNA is not detectable in the water used inthe water-drop step (lane b). The results demonstrate that DNA loss inthe washing step of the inventive method, although carried out undernon-binding conditions, is negligible.

Example 6: Yield of Plasmid DNA

DNA from E. coli cells carrying plasmid pCMVβ was prepared according tothe standard protocol described in example 2. In the final washing step(water-drop step), the incubation time was 10 seconds. After incubation,the particles were re-collected and eluted as described in example 2.

DNA from the eluate and from the water used in the water-drop washingstep was analyzed by agarose gel electrophoresis. The results are shownin FIG. 4. A strong plasmid DNA band is visible for the product(“elution”, right lanes). A faint DNA band is also visible in the waterused in the water-drop step (“water-drop”, left lanes). The resultsdemonstrate that a minor loss of plasmid DNA may occur in the washingstep of the inventive method (water-drop step). These findings suggestthat DNA yield depends on the length of the nucleic acids, such thatshorter nucleic acids elute more easily from the silica magneticparticles. However, the absolute DNA loss is still low for plasmid DNAand thus the inventive method is applicable for producing excellentyields of highly pure plasmid DNA.

Example 7: Effect of Incubation Time and Mechanical Force on DNA Yield

DNA from E. coli cells carrying plasmid pCMVβ was prepared according tothe standard protocol described in example 2. In the final washing step(water-drop step), the incubation time was varied from 0 to 24 hours.During that time period the particles were not moved or agitated butallowed to lie at the bottom of the tube After incubation, the particleswere re-collected and eluted as described for example 2.

DNA from the eluate and from the water used in the water-drop washingstep was analyzed by agarose gel electrophoresis. The results are shownin FIG. 5. Strong plasmid DNA bands are visible for the isolated DNA(“B: Eluate”). For water used in the washing step, only very faint DNAbands were visible after 0 and 6 hours (“A: Water from water drop”). Theamount of DNA in water increased within 24 hours and reached significantlevels. Overall, DNA elution increased over incubation time, although nomechanical force was applied to wash nucleic acids away from particles.The results suggest that loss of DNA can be decreased in the inventiveprocess by reducing the incubation time.

In order to determine the effect of mechanical force and moderateheating in the elution step a further, i.e. second elution step wasapplied in which the magnetic particles were suspended for 3 minutes at50° C. in a shaker at 1400 rpm. Such conditions represent common elutionconditions which usually result in quantitative elution. Thereby, itcould be verified if and how much DNA had still remained bound to themagnetic particles after the first elution procedure. FIG. 5demonstrates that after the first elution procedure with rather moderateelution conditions still some DNA had remained bond to the magneticparticles. Yet, additionally applying mechanical force and moderateheating further releases the DNA from the magnetic particles (“C: Eluateafter second elution procedure with mechanical force and moderateheating”). The results suggest that strong mechanical forces and heatingshould be avoided in the inventive washing step in order to obtain ahigh yield. After incubation for an extended time up to 24 hours, theamount of DNA in the eluate decreased, indicating that the DNA shouldhave been quantitatively released.

Example 8 Comparison of Water-Rinse and Water-Drop for Plasmid DNA

DNA from E. coli cells carrying on one hand plasmid pCMVβ (about 7.2kbp) and on the other hand plasmid UC21 (about 2.7 kbp) was prepared inparallel according to the standard protocols described in examples 1 and2. In the final washing step (water-drop step), the incubation time was5 seconds. After incubation, the particles were re-collected and elutedas described in examples 1 and 2. The results are shown in FIG. 6.

Short nucleic acids are known to elute from magnetic particles at afaster rate than long nucleic acids. It is assumed that this is due tothe lower number of contact points between the nucleic acids and thebeads. Therefore, it could have been expected that for the indicatedplasmids the water-drop method of the present invention results in atremendous loss of nucleic acid. Yet, surprisingly the results in FIG. 6show that although the magnetic particles with the bound plasmid DNA aresuspended in the washing solution, the loss of nucleic acid is very lowand in a similar order of magnitude as is seen for the short and rathersuperficial rinsing procedure of the state of the art. Anyway, at thesame time the purity of the plasmid DNA obtained with the water-dropwashing procedure according to the present invention is increasedcompared to the rinsing procedure of the state of the art because due tothe suspension of the particles more contaminations are removed.Consequently, the inventive method results in an improved purity withoutfurther loss of nucleic acid than in the state of the art.

1. A method for isolating nucleic acid from a sample solution, whereinthe nucleic acid is separated from the sample solution by adsorption tomagnetic particles, followed by separation of the magnetic particleswith the nucleic acid adsorbed thereto from the sample solution byapplication of a magnetic field, followed by at least one washing step(w), comprising (w1) suspending the magnetic particles with the nucleicacid adsorbed thereto in a washing solution, which is an aqueoussolution, which has a total salt concentration below 100 mM and does notcomprise an organic solvent, (w2) incubating the magnetic particles withthe nucleic acid adsorbed thereto in the washing solution, and (w3)separating the magnetic particles with the nucleic acid adsorbed theretofrom the washing solution.
 2. The method of claim 1, wherein the nucleicacid is DNA, RNA or both, DNA and RNA, preferably DNA.
 3. The method ofat least one of the preceding claims, wherein the washing solution has atotal salt concentration below 25 mM.
 4. The method of at least one ofthe preceding claims, wherein the washing solution consists of water andoptionally salts.
 5. The method of at least one of the preceding claims,wherein washing step (w) is carried out for 5 minutes or less.
 6. Themethod of at least one of the preceding claims, wherein the nucleicacid, preferably DNA, has a chain length of at least 10 kb or kbprespectively.
 7. The method of at least one of claims 1 to 5, whereinthe nucleic acid, preferably DNA, has a chain length of less than 10 kbor kbp respectively, wherein washing step (w) is carried out for 2minutes or less.
 8. The method of at least one of the preceding claims,with one or more of the following characteristics being applied inwashing step (w): the magnetic particles with the nucleic acid boundthereto are not subjected to a mechanical force the magnetic particleswith the nucleic acid bound thereto are not subjected to a mechanicalforce exerted oscillating and/or horizontally, the magnetic particleswith the nucleic acid bound thereto are subjected to a mechanical forceexerted vertically, preferably to gravity, the magnetic particles withthe nucleic acid bound thereto are subjected to a magnetic force,preferably exerted vertically, the magnetic particles with the nucleicacid bound thereto are suspended in the washing solution withoutapplying a mechanical force the magnetic particles with the nucleic acidbound thereto are suspended in the washing solution without applying amechanical force exerted oscillating and/or horizontally the magneticparticles with the nucleic acid bound thereto are suspended in thewashing solution with applying a mechanical force exerted vertically,preferably gravity, the magnetic particles with the nucleic acid boundthereto are suspended in the washing solution with applying a magneticforce, preferably exerted vertically.
 9. The method of at least one ofthe preceding claims, wherein before washing step (w), the magneticparticles with the nucleic acid adsorbed thereto are washed in at leastone pre-washing step (p) with a washing solution, which is an aqueoussolution having a total salt concentration above 500 mM and/or comprisesan organic solvent.
 10. The method of at least one of the precedingclaims, wherein after washing step (w) the nucleic acid is eluted fromthe magnetic particles with an elution solution.
 11. The method of atleast one of the preceding claims, wherein the magnetic particlescomprise silica.
 12. The method of at least one of the preceding claims,wherein the sample solution comprises a chaotropic salt and/or analcohol.
 13. The method of at least one of the preceding claims,comprising the steps of (a) adding magnetic particles to a samplesolution comprising the nucleic acid, (b) incubating the sample solutionfrom step (a) to allow adsorption of the nucleic acid to the magneticparticles, (c) applying a magnetic field to the sample solution fromstep (b) to fixate the magnetic particles with the nucleic acid adsorbedthereto, (d) removing the sample solution, (e) washing the magneticparticles with a washing solution in a washing step (w) of claim 1, and(f) optionally eluting the nucleic acid from said magnetic particleswith an elution solution.
 14. The method of at least one of thepreceding claims, wherein the method is an automated process.
 15. Use ofan aqueous solution, which has a total salt concentration below 100 mMand does not comprise an organic solvent, as a washing solution forwashing magnetic particles with nucleic acid adsorbed thereto in amethod for isolating nucleic acid from a sample solution, wherein themagnetic particles with the nucleic acid adsorbed thereto are suspendedin the washing solution.